Our goal remains to understand pancreatic beta-cell intermediary and energy metabolism in fuel stimulated insulin secretion and to explore how these processes are modified by neuro-endocrine factors. The focus is on glucose, glutamine and fatty acid metabolism, on the inter connections between the different metabolic pathways and on the fuel dependence of acetylcholine and GLP-1 stimulation of beta-cells. This program continues to be strongly influenced by biochemical genetic information about monogenic hypo- and hyperglycemia syndromes in man, linked to glucokinase, glutamate dehyrogenase, short chain hydoxy acyl-CoA dehydrogenase and SUR-1/Kir6.2. Related pharmacological and therapeutic issues are also considered, e.g. GKAs, SUR-1 inhibitors and GLP-1. We have formulated the following specific projects for the next grant period: 1) Use enzyme kinetics, biophysical techniques including crystallography, pharmacological glucokinase activators, the glucokinase regulatory protein and cell biological approaches to contribute to the comprehensive characterization of normal glucokinase, of glucokinase redesigned by special mutations and of the close to 250 natural mutant glucokinases that cause hypo- or hyperglycemia in man with the twofold goal of explaining the clinical phenotypes and of deepening our understanding of the enzyme's functions including its established glucose sensor role. 2) Apply a broad gamut of approaches including a new method for respirometry, tracer techniques involving radioactive and heavy isotopes, phosphorus-, 13C- and proton NMR and measurements of intracellular calcium to study metabolic pathways of pancreatic islet cells during insulin secretion stimulated by glucose, amino acids and fatty acids (alone or in combination, and also when influenced by neuroendocrine factors) to help elucidate still elusive triggering and amplification mechanisms involved in insulin release. 3) Explore the biochemistry and physiological significance of glucokinase in gonadotropes and thyrotropes of the pituitary gland to help understand the extra-pancreatic and extra-hepatic role of the enzyme.